Protein biochips that can present many different proteins simultaneously will greatly accelerate research and development in medical and biological sciences. One element essential for producing protein biochips is a fast and flexible method for immobilization of many different proteins. Methods that employ serial immobilization strategies using mechanical spotting devices suffer from numerous problematic issues and are constrained in their ability to produce one-of-a-kind chips. We have completed proof-of-concept demonstrations of a parallel immobilization strategy for producing protein biochips using self-assembling tags. Proteins are prepared by in vitro translation, which facilitates the laborious task of purifying thousands of different proteins. The in vitro translation techniques described here produce proteins with tags affixed at random positions, therefore, the resulting ensemble of immobilized proteins presents random orientations. This greatly improves interrogating the activity of the proteins. Proof-of-principle experiments have demonstrated that this scheme is effective for specific immobilization and detection of proteins. The objective of the work described in this proposal is to extend these preliminary experiments and to demonstrate that multiple proteins can be translated, spatially multiplexed by self-assembly on a biochip, and used for functional analysis. Automation is facile and will enable biochips with theoretically unlimited numbers of functional proteins for extensive commercial use. The proposed research concerns the development of a method to selectively bind ten (as a demonstration of feasibility) different synthetic proteins to multiple predetermined sites of a CombiMatrix biochip. The previously developed Combimatix biochip is an addressable microelectrode array which, allows the synthesis of various predetermined oligonucleotides at specific electrode sites through a sequence of electrochemical/chemical reactions. By tagging desired proteins with oligonucleotides (via, eg. Streptavidin/Biotin) the protein can be attached at an electrode site, which was used to synthesize the complementary oligonucleotide. The result is a biochip, which has a variety of known proteins immobilized in known array positions. PROPOSED COMMERCIAL APPLICATION: The proposed research is directed to develop a method of manufacture of custom protein microchips in a fast, efficient, automatic and inexpensive way. These chips can be used for diagnostic analysis, drug-lead screening, and functional genomics studies including protein-protein and protein-target interactions, catalytic and inhibitory activity.